The ability of antibodies to recognise specific antigens has made them highly useful and effective tools in medicine and biotechnology. Antibodies specific for antigens on selected types of cell have been used to target these antibodies to the selected cell. The binding of antibodies to receptors on cells has been found in some cases to affect the function of the cell. Therapeutic and diagnostic agents have also been conjugated to antibodies to specifically target these agents to selected cells. This technique has been used particularly in the targeting of cancer cells. Antibodies have also been used to target antigens on virally- or bacterially-infected cells, to target other molecules, such as TNFα, or for use in assays. In biotechnology, antibodies have many uses such as probes, in purification and in catalysis.
All whole antibody molecules consist of four polypeptide chains—two identical heavy chains and two identical light chains. Each chain comprises both variable and constant domains. Light chains comprise two domains: VL and CL, whilst heavy chains comprise at least five domains: VH, CH1, hinge, CH2 and CH3 and an optional CH4. The four chains are always organised in the same general fashion: the two heavy chains are linked together by at least one disulphide bond and each heavy chain is also linked to one of the light chains by a disulphide bond such that both light chains are linked to a separate heavy chain.
Whole antibody molecules are roughly Y-shaped and consist essentially of two main functional parts. The first functional part is responsible for the recognition of specific antigens and is formed by the upper part of the arms of the Y. The antigen binding region in each part comprises one VH domain and one VL domain. Each variable domain contains three hypervariable regions which, together with the three hypervariable regions in the other chain, form the antigen-binding site. These hypervariable regions are known as complementarity determining regions (CDR1, CDR2 and CDR3). The CDRs, which form loops, are supported on framework regions. Due to its variability between antibodies, the regions comprising VH and VL are known as the ‘V’ regions.
The second functional part is responsible for triggering the effector functions of other cells that will dispose of the antigen recognised by the antibody and is formed by the lower parts of the arms and the stem of the Y. This region is known as the constant ‘C’ region due to its relative constancy. It comprises the CL domains and all the heavy chain C domains.
There are two types of light chain: κ and λ, and five types of heavy chain: α, δ, γ, ε and μ. The class of the antibody is determined by the type of heavy chain it has: IgA, IgD, IgE, IgG and IgM respectively.
Antibody fragments, which have had part of their constant region removed by enzymatic cleavage, are also used in medicine and biotechnology. These include Fab, Fab′, F(ab′)2 and Fv fragments.
It is known to direct production of large amounts of monoclonal antibodies (having a particular antigen-specificity) by fusing an antibody-producing spleen cell with a myeloma cell, resulting in a hybridoma (Kohler, G. and Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256, 495-497 (1975)). However, such antibodies are unsuitable for use in human therapy as they are immunogenic in humans. Most monoclonal antibodies are produced by non-human cells.
Recombinant DNA techniques have been developed which enable the useful properties of more than one antibody to be combined to make one new antibody. The production of chimeric antibodies, in which the antigen-binding site comprising the complete V region of one antibody is linked to the constant region from a different antibody, is described in EP-A-0120694 (Celltech Limited), EP-A-0125023 (Genentech Inc. and City of Hope), EP-A-0171496 (Research Development Corporation, Japan), EP-A-0173494 (Stanford University) and WO-A-86/01533 (Celltech Limited).
WO-A-86/01533, for example, describes the preparation of a chimeric antibody in which murine V regions are joined to human constant regions. However, the large proportion of residues in chimeric non-human/human antibodies which are derived from the non-human donor result in the possibility of the antibody eliciting a potentially harmful immunological response, particularly if administered over a prolonged period (Begent et al., Br. J. Cancer, 62, 487 (1990)).
Antibody ‘humanisation’ is a technique which makes non-human/human chimeric antibodies appear more like human antibodies to the human immune system and has been developed in an attempt to overcome the unwanted immunological response mentioned above. EP-A-0239400 (Winter) describes how, instead of using a complete murine variable region, the CDRs of a murine monoclonal antibody are grafted onto the framework regions of the variable domains of a human antibody. Thus, it is only the CDRs forming the antigen-binding domain itself that are murine and the other residues are human.
Reichmann et al., (“Reshaping human anitbodies for therapy”, Nature, 332, 323-324, 1988) found it to be advantageous to convert other human residues in the variable domain to their non-human donor counterparts to improve antigen-binding activity. Such a residue was found at position 27 of the human heavy chain, which, when converted from the human serine to the corresponding rat residue (phenylalanine), resulted in improved antigen-binding ability. Another such residue was found at position 30. However, a construct which contained a human serine to rat tyrosine change at position 30 of the heavy chain in addition to the change at position 27 mentioned above, did not have a significantly altered binding activity over the humanised antibody with the serine to phenylalanine change at position 27 alone.
Heavy chain residues 27 and 30 are within the structural loop adjacent to CDR1. Queen et al., (WO 90/07861) conjectured that other residues which interact with the CDRs are also important in determining antigen-binding affinity. With this in mind, Queen et al., proposed four criteria for determining which residues should come from the donor and which from the acceptor when designing humanised antibodies. In a more definitive analysis, Adair et al., (WO 91/09967) disclosed a hierarchy of residue numbers that will enable a humanised antibody to be designed.
A number of reviews discussing CDR-grafted antibodies have been published including Vaughan et al., (Nature Biotechnology, 16, 535-539, 1998).
Antibody conjugates, in which the constant region has been fused to effector or reporter molecules which may act as therapeutic or diagnostic agents, have also been described (WO 95/01155, U.S. Pat. Nos. 3,927,193, 4,331,647, 4,348,376, 4,361,544, 468,457, 4,444,744, 4,460,459 and U.S. Pat. No. 4,460,561 and reviews by Waldmann, T. A., Science, 252, 1657, (1991); Koppel, G. A., Bioconjug. Chem., 1, 13, (1990); Oeltmann, T. N. and Frankel, A. E., FASEB J., 5, 2334, (1991); and van den Bergh, H. E., Chemistry in Britain, May 1986, 430-439).
It was known to produce normal, chimeric or humanised antibodies by transfecting a suitable host cell with two expression vectors, one containing a DNA sequence encoding the heavy chain and one containing a DNA sequence encoding the light chain of the required antibody (WO-A-91/09967). Alternatively, it was known to transfect a suitable host cell with an expression vector that contains both the DNA sequence encoding the heavy chain and the DNA sequence encoding the light chain of the required antibody. In the latter example, the DNA sequences encoding the heavy chain and the light chain are either under the control of their own individual promoters (WO-A-91/09967) or are present in a dicistronic message (Better, M., Paul Chang, C., Robinson, R. R. and Horwitz, A. H. Escherichia coli: Secretion of an Active Chimeric Antibody Fragment. Science, 240, 1041-1043 (1988)).
A dicistronic message was used by Better et al., to produce a chimeric mouse L6 Fab antibody directed towards a ganglioside antigen expressed on the surface of many human carcinomas. A dicistronic message was chosen in this case in an attempt to ensure that both the truncated heavy chains (Fd) and the κ light chains were translated in close physical proximity so that they would assemble correctly and be secreted. Dicistronic messages are only able to function in bacteria whereas the ‘one gene, one promoter’ concept functions in both mammals and bacteria. In a dicistronic message, a promoter is associated only with the first cistron. The second cistron is transcribed by the polymerase ‘reading through’ to the second cistron such that both cistrons are represented by a single RNA molecule. The two coding DNA cistrons are separated by a stretch of DNA known as an ‘intergenic sequence’ or ‘IGS’. This IGS region is also present in the RNA molecule that is transcribed from the DNA.
Optimisation of the translational initiation rate has for some time been recognised as essential for high level expression of secreted heterologous proteins in E. coli (Simmons, L. and Yansura, D. Nature Biotech, 14, 629-634 (1996)). However, different Fab's have different framework and CDR sequences which confer different properties to the molecule, including differences in the ease (or rate) of folding within the E. coli periplasm (Knappik, A. and Pluckthun, A. Prot Eng 8, 81-89 (1995)). For example, a Fab′ which folds into its native conformation easily and rapidly within the E. coli periplasm is likely to be ‘tolerated’ at a high level and rapid translation can be accommodated to achieve high-level accumulation. A Fab′ which folds more poorly is likely to be tolerated less well by the host bacterium if rapidly translated, potentially saturating host folding/secretion machinery and having a deleterious effect on host cell physiology.
Thus, when producing antibodies using a host cell-expression vector system, a high level of expression of a particular antibody may be tolerated, but for a different antibody, a high level of expression might prove toxic to the cell, perhaps because of different efficiencies of secretion or folding.
The level of expression of a particular antibody is determined by the amount of heavy and light chains present. For maximal production, the ratio of heavy chains to light chains should be balanced, such as equal quantities of both. However, if either the heavy or the light chain is present in a lesser amount, this will limit the amount of antibody produced. Accumulation of excess heavy chain is likely to be particularly poorly tolerated.
In the case of an antibody encoded by a dicistronic message, the upstream cistron may contain either the DNA coding for the heavy chain or the light chain of an antibody with a particular antigen specificity. The downstream cistron would then encode the respective light chain or heavy chain partner. It would be advantageous if the level of expression of the antibody chain corresponding to the coding DNA in the downstream cistron of a dicistronic message could be regulated to produce the desired level of expression for a particular antibody.